Viuff D, Hyttel P, Avery B, Vajta G, Greve T, Callesen H, Thomsen P D
Department of Clinical Studies, Reproduction, Agricultural University, DK-1879 Frederiksberg C, Denmark.
Biol Reprod. 1998 Sep;59(3):626-31. doi: 10.1095/biolreprod59.3.626.
Ribosomal RNA, rRNA genes, and silver-staining nucleolar proteins were visualized in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst using a sequential fluorescent in situ hybridization (FISH) and a silver-staining procedure. At FISH, the rRNA was differentiated from the signal of the rRNA genes through comparison of RNase- and non-RNase-treated embryos. Both RNase- and non-RNase-treated 2-cell embryos revealed up to 10 small clusters of fluorescein isothiocynate (FITC) labeling in interphase nuclei. The RNase-treated 4-cell embryos displayed the same FITC pattern as the 2-cell embryos. In the non-RNase-treated 4-cell embryos, in contrast, the clusters were larger and included numerous small spots. In 2-cell as well as 4-cell embryos, almost all FITC-labeled clusters colocalized with silver-stained spots. In the RNase-treated 8- to 16-cell embryos, up to 10 clusters of FITC labeling were organized as one or more large spots surrounding a central faint but homogeneously labeled area. The non-RNase-treated 8- to 16-cell embryos displayed similar complexes, but the central areas consisted of small labeled spots. In 8- to 16-cell embryos, all FITC-labeled clusters were again colocalized with silver-stained areas. In the blastocysts, 1-6 big clusters of FITC labeling colocalized with silver staining. In the RNase-treated blastocysts, the FITC labeling was typically located at the edges of the silver-stained areas, whereas in the non-RNase-treated blastocysts, the FITC labeling totally covered the silver-stained areas. In conclusion, there is a close association between the rRNA genes and silver-staining nucleolar proteins in in vitro-produced bovine embryos from the second cell cycle, i.e., the 2-cell stage; the first rRNA is apparently transcribed during the third cell cycle, and during the fourth cell cycle the molecular composition of functional nucleoli is established.
利用连续荧光原位杂交（FISH）和银染程序，在体外生产的从2细胞期到囊胚期的牛胚胎中观察核糖体RNA、rRNA基因和银染核仁蛋白。在FISH实验中，通过比较核糖核酸酶处理和未处理的胚胎，将rRNA与rRNA基因的信号区分开来。核糖核酸酶处理和未处理的2细胞胚胎在间期细胞核中均显示出多达10个小的异硫氰酸荧光素（FITC）标记簇。核糖核酸酶处理的4细胞胚胎显示出与2细胞胚胎相同的FITC模式。相比之下，未用核糖核酸酶处理的4细胞胚胎中，这些簇更大且包含许多小斑点。在2细胞和4细胞胚胎中，几乎所有FITC标记的簇都与银染斑点共定位。在核糖核酸酶处理的8至16细胞胚胎中，多达10个FITC标记簇组织成围绕中央微弱但均匀标记区域的一个或多个大斑点。未用核糖核酸酶处理的8至16细胞胚胎显示出类似的复合体，但中央区域由小的标记斑点组成。在8至16细胞胚胎中，所有FITC标记的簇再次与银染区域共定位。在囊胚中，1至6个大的FITC标记簇与银染共定位。在核糖核酸酶处理的囊胚中，FITC标记通常位于银染区域的边缘，而在未用核糖核酸酶处理的囊胚中，FITC标记完全覆盖银染区域。总之，在体外生产的牛胚胎中，从第二个细胞周期即2细胞期开始，rRNA基因与银染核仁蛋白之间存在密切关联；第一个rRNA显然在第三个细胞周期转录，并且在第四个细胞周期建立功能性核仁的分子组成。
